Colonic motility results from coordinated contractions of the muscles that line the colon wall. Aberrant contractions lead to a variety of motility disorders including irritable bowel syndrome, diarrhea, and constipation. In this project we will study the factors that couple excitation to contraction and regulate colonic motility. Much of the calcium (Ca2+) needed to trigger excitation-contraction coupling appears to enter colonic smooth muscle cells through voltage-dependent Ca2+ channels. Previous work has suggested that activation of these channels provides a mechanism for excitability in colonic muscles, thus, directly linking electrical excitation to contraction. In the proposed study changes in intracellular Ca2+ ([Ca2+]i) in response to the electrical activities of circular and longitudinal muscles will be characterized in intact muscles, and the relationship between Ca2+ current and [Ca2+]i will be studied in isolated cells. Factors that regulate voltage- dependent Ca2+ current and [Ca2+]i; ii) the release of Ca2+ from intracellular stores; and iii) entry of Ca2+ through transporters or channels other than L-type Ca2+ channels will be studied. Recent evidence demonstrates the presence of non-selective cation channels in colonic muscles. These channels are activated by cholinergic and peptidergic stimulation. The properties of this conductance will be characterized, and the possibility that Ca2+ might enter cells via this pathway will be explored. Factors that regulate Ca2+ clearance from cells after activation will also be investigated. The expression and role of Na/Ca exchange and Ca2+ ATPases in the clearance of Ca2+ will be characterized with molecular and electrophysiological techniques. The role of the sarcoplasmic reticulum in the clearance of Ca2+ will also be studied. Recent evidence has suggested that regulation of the sensitivity of the contractile apparatus to Ca2+ may also link excitation to contraction. Therefore, we will study the relationship between force and [Ca2+]i in response to physiologically relevant agonists such as neurotransmitters, hormones and paracrine substances. We will also test whether application of multiple excitatory agonists causes synergistic responses, perhaps by increasing the sensitivity of the contractile apparatus to Ca2+. The relationship between [Ca2+]i and myosin light chain phosphorylation will also be characterized. This project will provide comprehensive information about the cellular factors that regulate contractile force in colonic muscles.